PECAM-1 null marrow exhibited an expanded hematopoietic stem cell population and increased quiescent G₀ phase cells. (A-C) Representative graphs of flow cytometric assays on the hematopoietic stem cell populations of WT, Het, and CD31KO cells (Lin⁻, Sca-1⁺, c-kit⁺ cells [LSK]). The same number of BM mononuclear cells (250 000) was used in each FACS assay. The top panels are representative FACS plots of Lin⁻ population gating. The shaded areas of these graphs represent gated Lin⁻ areas, which exclude high-intensity, intermediate-intensity, and low-intensity PerCP-positive populations. Nonstained controls (red lines) were included in these graphs. The bottom panels are corresponding representative graphs of the percentages of Sca-1⁺, c-kit⁺ cells in gated Lin⁻ populations of WT, Het, and CD31 KO littermates. (D) The percentage of LSK cells in BM fraction (LSK cell number divided by BM cell fraction number) was significantly increased in KO marrow (denoted in the heavy outlined quadrants in panels A-C and quantitated in panel D; n = 3). (E) Distribution of cells in G₀ versus G₁ in the Lin⁻ BM mononuclear WT, Het, and CD31KO cell populations indicates increased CD31KO hematopoietic progenitor cells were arrested in quiescent G₀ phase (n = 3). Marrow cells were stained with lineage antibodies, Pyronin Y(RNA dye), and Hoechst 33342 (DNA dye). Lin⁻ cells that had low Hoechst staining (cells in G₀/G₁ phase) were gated for analyses. The average G₀ percentage in Lin⁻Hoechst^low cells from CD31KO marrow is significantly increased compared with that of WT and Het marrows (n = 3). (F) The mean percentages of annexin V-positive, PI-negative cells representing apoptotic cells were not significantly different in WT, Het, and CD31KO Lin⁻, c-kit⁺ populations (n = 3). Vertical lines represent standard deviations.